Study On The Role And Mechanism Of Circ₀006528 Inpromoting Human Breast Cancer Proliferation,Invasion And Migration

Objective To investigate the role and mechanism of circ₀006528 on proliferation,migration and invasion of human breast cancer.Methods1.qRT-PCR was used to detect the expression levels of circ₀006528 in 97 cases of human breast cancer tissues,and analyzed the relationship between circ₀006528 expression and clinicopathological characteristics and prognosis.2.circ₀006528 small interfering RNA?siRNA?was designed to transfect into human breast cancer cell lines BT-549 and ZR-75-30 with higher circ₀006528 expression;The circ₀006528 overexpression plasmid was constructed and transfected into human breast cancer cell lines MDA-MB-231 and Hs578 T with lower circ₀006528 expression,and empty vector transfection group was used as control group.3.BT-549 cells were transfected with circ₀006528 siRNA and/or miR-7-5p inhibitor to down-regulate circ₀006528?KD group?and/or miR-7-5p expression?inhibitor group?,and the empty vector transfection groups were used as control groups respectively.The treatment groups were divided into: KD-NC +miR-NC?mock group?;KD +miR-NC?KD group?;KD-NC + miR-7-5p inhibitor?miR-7-5p inhibitor group?;KD + miR-7-5p inhibitor.MDA-MB-231 cells were transfected with overexpressing plasmid and/or miR-7-5p mimic separately to up-regulate circ₀006528?OE group?and/or miR-7-5p expression?mimic group?,and the empty vector transfection groups were used as control groups respectively.The treatment groups were divided into: OE-NC+ miR-NC?mock group?;OE+ miR-NC?OE group?;OE–NC+ miR-7-5p mimic?miR-7-5p mimic group?;OE+miR-7-5p mimic.4.EdU incorporation experiment,CCK8 assay,flow cytometry,transwell cell invasion and migration experiments,scratch assay were used to detect DNA synthesis,proliferation,cell cycle,apoptosis,invasion and migration levels in each group,respectively.Western-blot was used to detect Raf1 protein expression and the phosphorylation status of MEK1/2 and ERK1/2.Luciferase reporter assay was used to detect the direct interaction between circ₀006528 and miR-7-5p.Results1.The result of qRT-PCR showed that the expression of circ₀006528 was significantly higher in human breast cancer tissues than in adjacent nontumorous tissues,and the difference was statistically significant?P<0.01?;compared with the breast cancer in stage I,there was a significantly higher circ₀006528 expression in stage III breast cancer tissues,and the difference was statistically significant?P<0.05?;Kaplan-Meier survival analysis showed that the higher circ₀006528 expression in breast cancer patients was corresponding with the shorter relapse-free survival rate and overall survival rate,and the difference was statistically significant?P<0.05?.It is suggested that high circ₀006528 expression in human breast cancer tissues was related to the TNM stages?tumor-node-metastasis stages?and prognosis of breast cancer.2.The DNA synthesis,proliferation,migration and invasion levels were significantly decreased,and induced G2 phase arrested and cell apoptosis when knocked-down the expression of circ₀006528 in human breast cancer cell lines BT-549 and ZR-75-30 compared with the control groups.The DNA synthesis,proliferation,migration and invasion levels were significantly increased and apoptosis rates were decreased when over-expressed circ₀006528 in human breast cancer cell lines MDA-MB-231 and Hs578 T compared with the NC groups,and the differences were statistically significant?P<0.05?.It is suggested that high expression of circ₀006528 could promote the DNA synthesis,proliferation,migration,invasion,and inhibit cell apoptosis in breast cancer cells.3.The luciferase reporter experiment showed that the inhibitory rate of luciferase activity in the wild type circ₀006528 was 63% compared with the control group,but the mutation and the circ₀006528 of the miR-7-5p binding site had no effect,which proved the direct effect of circ₀006528 and miR-7-5p.When the circ₀006528 was mutated with miR-7-5p binding sites,the mutated circ₀006528 had no inhibitory effect on luciferase activity,which verified the direct effect of circ₀006528 and miR-7-5p.4.The functional rescue experiments showed that down-regulation of miR-7-5p promoted DNA synthesis,proliferation,migration and invasion,Raf1 protein expression and MEK1/2 and ERK1/2 phosphorylation,which were partially reversed by circ₀006528 siRNA in BT-549 cells.The differences were statistically significant?P<0.05?.Overexpression of mi R-7-5p leading to the inhibition of cell DNA synthesis,proliferation,migration and invasion,Raf1 protein expression and MEK1/2 and ERK1/2 phosphorylation,and this effect was partially reversed by circ₀006528 in MDA-MB-231 cells.The differences were statistically significant?P<0.05?.Conclusions1.Circ₀006528 was higher expressed in breast cancer tissues than those in adjacent non-tumous tissues.The high circ₀006528 expression was related to TNM stages and shorter relapse-free survival and overall survival time.2.The high expression of circ₀006528 promotes the DNA synthesis,proliferation,migration and invasion,and inhibits cell apoptosis in human breast cancer cells.3.Circ₀006528 mediates the growth and metastasis through circ₀006528 / miR-7-5p/Raf1/MAPK/ERK signaling pathway in breast cancer.We have identified a new carcinogenic circRNA—circ₀006528 in breast cancer,and elucidated its role and molecular mechanism in the progression of breast cancer.These findings suggest that circ₀006528 might act as a tumor stimulative factor and may be a potential strategy to reduce the recurrence and metastasis of breast cancer.